Abstract
Normally, isothermal titration calorimetry (ITC) is used to study binding reactions between two different biomolecules. Self-association processes leading to homo-oligomeric complexes have usually not been studied by ITC; instead, methods such as spectroscopy and analytical ultracentrifugation, which only provide affinity and Gibbs-free energy (i.e., K(D) and DeltaG), are employed. We here demonstrate that complete thermodynamic descriptions (i.e., K(D), DeltaG, DeltaH, and DeltaS) for self-associating systems can be obtained by ITC-dilution experiments upon proper analysis. We use this approach to probe the dissociation (and thus association) equilibrium for the heptameric co-chaperonin proteins 10 (cpn10) from Aquifex aeolicus (Aacpn10-del25) and human mitochondria (hmcpn10). We find that the midpoints for the heptamer-monomer equilibrium occur at 0.51 +/- 0.03 microM and 3.5 +/- 0.1 microM total monomer concentration (25 degrees C), for Aacpn10-del25 and hmcpn10, respectively. For both proteins, association involves endothermic enthalpy and positive entropy changes; thus, the reactions are driven by the entropy increase. This is in accord with the release of ordered water molecules and, for the thermophilic variant, a relaxation of monomer-tertiary structure when the heptamers form.